A risk factor for several neurodegenerative diseases is inheritance of the epsilon 4 allele of apolipoprotein E (APOE). The best established increased risk with inheritance of epsilon 4 allele of APOE is for Alzheimer's disease (AD) where inheritance of the epsilon 2 allele also appears to be protective. Although the subject of much interest and study, the mechanism behind these differing risks remains to be fully explained. We hypothesize that one mechanism for the difference in risk of AD is due to isoform-specific differences in apoE-associated proteins. The recently developed approach of high-throughput quantitative proteomic analysis combining isotope-coded affinity tagging (ICAT) with peptide analysis by LC/MS-MS allows us to identify and compare the relative abundance of apoE-associated proteins from different sources. In this study, I will affinity purify apoE-associated proteins from 5 human brain regions and compare the composition and relative abundance of each apoE-associated protein among different APOE genotypes in elderly individuals without dementia, and between AD patients and control individuals of the same genotype. In addition, I will perform identical analyses for cerebral homogenates and synaptosomes prepared from young and old apoE targeted replacement mice expressing human apoE isoforms under the mouse apoE promoter. In each case I will (i) identify proteins by proteomic analysis of pooled tissue samples, (ii) confirm the presence of selected proteins by Western blot of the same pooled samples, and (iii) statistically validate these differences by Western blotting of individual samples, as well as further validation by immunohistochemistry. I have contributed to recent studies that have successfully employed this three-step approach to identify proteins that are elevated in lumbar cerebrospinal fluid from probable AD patients and proteins that are present in neurofibrillary tangles, and now propose to use this novel technology to investigate apoE isoforms. Completion of this study of apoE-associated proteins will identify proteins that may be contributing to apoE isoform-specific risk or protection and provide candidate proteins to follow up with more detailed mechanistic studies in models of neurodegenerative diseases. [unreadable] [unreadable]